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  • Size Selection of Adaptor-ligated DNA (E7445)

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     This caution sign signifies a step in the protocol that has multiple paths leading to the same end point but is dependent on a user variable, like the amount of input DNA.

    Protocol

    Note: Using Agencourt AMPure XP Beads for size selection of adaptor-ligated DNA is strongly recommended.

    Method 1: Size Selection Using AMPure XP Beads

     Caution: The following size selection protocol is for libraries with 200 bp inserts only. For libraries with larger fragment inserts, please optimize bead: DNA ratio accordingly.

    1. To the ligation reaction (83.5 μl), add 16.5 μl nuclease-free water to bring the reaction volume to 100 μl.
      Note: X refers to the original sample volume of 100 μl from the above step.
    2. Add 60 μl (0.6X) resuspended AMPure XP beads and mix well on a vortex mixer or by pipetting up and down at least 10 times.
    3. Incubate for 5 minutes at room temperature.
    4. Quickly spin the tube in a microcentrifuge and place the tube on an appropriate magnetic rack to separate beads from the supernatant. After the solution is clear (about 5 minutes), carefully transfer the supernatant containing your DNA to a new tube (Caution: do not discard the supernatant). Discard beads that contain the unwanted large fragments.
    5. Add 25 μl (0.25X) resuspended AMPure XP beads to the supernatant (160 μl), mix well and incubate for 5 minutes at room temperature.
    6. Quickly spin the tube in a microcentrifuge and place the tube on an appropriate magnetic rack to separate beads from the supernatant. After the solution is clear (about 5 minutes), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain the desired DNA targets (Caution: do not discard beads).
    7. Add 200 μl of freshly prepared 80% ethanol to the tube while in the magnetic rack. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant.
    8. Repeat Step 7 once. 
    9. Briefly spin the tube, and put the tube back in the magnetic rack.
    10. Completely remove the residual ethanol, and air dry beads for 10 minutes while the tube is on the magnetic rack with the lid open.
    11. Elute DNA target from the beads with 25 μl nuclease-free water. Mix well on a vortex mixer or by pipetting up and down, then put the tube in the magnetic rack until the solution is clear.
    12. Without disturbing the bead pellet, transfer 20 μl of the supernatant to a clean PCR tube, and proceed to PCR enrichment.
      Note: Be sure not to transfer any beads. Trace amounts of bead carry over may affect the optimal performance of the polymerase used in the NEBNext High-Fidelity 2X PCR Master Mix in the subsequent PCR step.


    Method 2: Size Selection Using an E-Gel® SizeSelect™ Gel (Life Technologies, Inc.)

    1. Purify the adaptor-ligated DNA using QIAGEN MinElute® PCR Purification Kit and elute in 12 μl of nuclease free water.
    2. Perform size selection using E-Gel.
    3. Purify DNA sample on one QIAquick column and elute in 25 μl of nuclease free water.
    4. Transfer 20 μl of the supernatant to a clean PCR tube, and proceed to PCR enrichment.


    Method 3: Size Selection Using a 2% Agarose Gel

    1. Purify the adaptor-ligated DNA using QIAGEN MinElute PCR Purification Kit and elute in 12 μl of nuclease free water.
      IMPORTANT: Before eluting the DNA from the column, centrifuge the column with the lid of the spin column open for 5 minutes at 13,200 rpm. Centrifugation with the lid open ensures that no ethanol is carried over during DNA elution. Residual ethanol interferes with the correct loading of the sample on the gel.
    2. Pour a 5 mm thick 2% agarose gel with a 1 mm thick gel space and allow to cool.
    3. Run adaptor ligated cDNA for 2 hours at 150 V or until the dye front has traveled 10 cm.
    4. Isolate the desired cDNA fragment from the gel.
    5. Purify DNA from the gel slice using a QIAquick® Gel Extraction Purification Kit, purifying the sample on one column and elute in 25 μl of nuclease free water.
    6. Transfer 20 μl of the supernatant to a clean PCR tube, and proceed to PCR enrichment.